Immunogenicity in the past year we have succeeded in identifying the major B cell epitopes recognized by the human immune system and have made a new immunotoxin, HA22-LR-010, that is highly active and whose reactivity with human anti-sera is greatly reduced. We are trying to further decrease reactivity by making further mutations in the new immunotoxin. We have also initiated a program to identify and remove human specific T cell epitopes. We have now identified all the major T cell epitopes in the toxin and using alanine scanning mutagenisis identified which amino acids within the epitope can be mutated to silence the epitope and maintain high cytotoxic activity. Using this information we have made 2 new types of recombinant immunotoxins. One of these has all the major T cell epitopes silenced. The other has a combination of mutations that remove B and T cell epitopes. These new RITs are now being tested in mice with human tumor xenographs. Based on our ability to remove B cell epitopes, Roche has produced a new immunotoxin targeting mesothelin which is now in preclinical development. In collaboration with David FitzGerald we have begun to study how protein synthesis arrest leads to apoptosis and have identified BAK, Bax, MCL-1 and BCL-XL as critical players in this process. We are also studying how protein phosphorylation regulates sensitivity of cells to immunotoxin mediated cell death by doing knock down studies of tyrosine and serine/threonine kinases and measuring the effect of knock down of these kinases on immunotoxin mediated cell death. We have identified the insulin receptor, HCK, BMX and the PDGF beta receptor as kinases that regulate immunotoxin killing. Lowing levels of these proteins enhances immunotoxin action. We have also identified several TK inhibitors that enhance immunotoxin action against cultured tumors cells and tumors growing in mice.